Figure 3

Orc proteins cosediment mainly with topo II. (A) The pelleted chromatin in MN-treated nuclei (Fig. 2) was partially suspended in a low salt – high EDTA buffer and investigated by centrifugation through a sucrose gradient (5 – 20%) made up in the same buffer. A part of each gradient fraction was deproteinized and analyzed by agarose gel electrophoresis and staining with ethidium bromide (upper panel). Another part of each fraction was investigated by western blotting. (B) The combined fractions 8 and 9 in (A) were incubated with either control antibodies (IgG) or antibodies specific for Orc2p or topo II. The precipitates were rigorously washed with 80 mM NaCl in NE buffer. The supernatants and the immunoprecipitates were analyzed by western blotting.